Prior to the present invention, methods for determining hydrolytic analytes included chromogenic esters which, when hydrolyzed by esterase or protease, produced a colored alcoholic product, the intact ester being of a different color from the free alcohol. Many of these systems included accelerator compounds and diazonium salt coupling agents.
2.1 Chromogenic Esters
Thus, there exists in the prior art a body of references which disclose the use of certain esters which, when cleaved by enzymatic activity, result in the formation of color or other detectable species. British Pat. No. 1,128,371 discloses the use of indoxyl and thioindoxyl esters as useful chromogens in detecting hydrolytic enzymes in body fluids. The enzymes cleave the ester to generate free indoxyl, which subsequently oxidizes to form the dimeric product indigo, a readily observable blue dye. Such activity is said to be due to, among other enzymes, cholinesterase. This patent also teaches that, in addition to the indoxyl portion of the ester substrate, the acid radical is chosen with particular reference to the enzyme to be detected. For example, it is stated that the acid radical can be acetate, laurate or stearate for detection of esterase or lipase, respectively. For detecting enzymes such as phosphatase or sulfatase the acyl radical can be inorganic. Thus, the British Patent teaches the use of chromogenic esters as substrates for determining esterolytic enzymes, such esters comprising indoxyl or thioindoxyl as the alcoholic moiety of the ester, the acyl moiety being tailored to the particular enzyme to be determined.
The affect of careful acyl radical selection is nowhere more clearly exemplified than in two references which demonstrate esterase specificity for esters in which the acyl radical comprises an N-protected amino acid or peptide. Thus Janoff, et al., Proc. Soc. Exper. Biol. Med. 136: 1045-1049 (1971) teaches that alanine esters are specific substrates for esterase obtained from human leukocytes. Specifically, this reference teaches that an extract of human leukocyte granules is capable of hydrolyzing n-acetyl-L-alanyl-L-alanyl-L-alanine methyl ester. Moreover, L-alanine-p-nitrophenyl ester was similarly hydrolyzed to yield the yellow p-nitrophenol colorform.
Similarly, Sweetman et al., Jour. Hist. Soc., 22:327-339 teaches the use of 1-naphthyl N-acetyl-DL-alanine, 1-naphthyl N-acetyl-L-alanyl-L-alanyl-L-alanine and 1-naphthyl butyrate to demonstrate the presence of esterase.
U.S. Pat. No. 4,278,763, assigned to Boehringer Mannheim GmbH, combines these teachings in arriving at the indoxyl or thioindoxyl esters of amino acids or peptides as still another example of a traditional colorogenic substrate for leukocytic esterase activity. Like the Janoff and Sweetman references, the Boehringer patent teaches the equivalence of protease and esterase in their esterolytic penchants.
2.2 Accelerators
It is known that ester hydrolysis reactions can be activated by the presence of many nucleophilic agents, including a myriad of alcohols. Thus, the rate of hydrolysis of phenyl acetate and p-nitrophenyl acetate by esterase is increased 2.5 to 5.5 times upon addition of methanol or butanol. Greenzaid and Jencks, Biochemistry, 10(7), 1210-1222 (1971). Moreover, the effect increases with the length of the n-alkyl group. Wynne and Shalatin, Eur. J. Biochem., 31: 554-560 (1972).
In particular, this activation affect of alcohols has been observed with esters of amino acids. p-Nitrophenyl-N-acetyl-L-alaninate hydrolysis is activated (accelerated) by the presence of methanol. Fastrez and Fersht, Biochemistry, 12(11), 2025-2034 (1973). High molecular weight alcohols increase the rate of esterase-induced hydrolysis of p-nitrophenyl-t-BOC-L-tyrosinate. Ashe and Zimmer, Biochem. and Biophys. Res. Comm., 75(1), 194-199 (1977). The disclosure of U.S. Pat. No. 4,299,917 describes other known ester hydrolysis activators such as certain metal complexes, pyridine derivatives and imidazoles.
2.3 Diazonium Salt Coupling Agents
Also known is the use of certain diazonium salts to couple with phenols and pseudophenols to produce azo dyes. Martinet and Dornier Compt. Rend., 170, 592 (1920). Such a technique is used in an esterase analysis whereby indoxyl esters are hydrolyzed via esterase to produce indoxyl, which is in turn coupled with a diazonium salt to form the corresponding azo dye. Holt and Hicks, J. Cell Biol. 29, 261-366 (1966); Gossrau, Histochemistry, 57, 323-342 (1978); West German Offenlegungschrift No. 30 17 721, filed May 9, 1980.
2.4 Summary
To summarize the background of technological developments leading up to the present invention, several methods are known for assaying hydrolytic enzymes and leukocyte cells in solution. For measuring leukocyte populatons, in urine, for example, microscopy has long been the preferred method. Thus, the technician was required to make a microscope slide of a urine sample and count the number of leukocyte cells in the field of view of a microscope; a procedure requiring an inordinate expenditure of time and expensive equipment such as a microscope and centrifuge.
Chemical and biochemical techniques are rapidly challenging the microscope for assaying leukocytes in diagnostics and are time-honored tools in the research laboratory. Chromogenic esters which have formed the keystone of chemical tests include the following alcoholic and acyl moieties:
______________________________________ Alcoholic (phenolic) Moieties Acyl Groups ______________________________________ indoxyl acetate thioindoxyl butyrate p-nitrophenyl laurate .alpha.-naphthol stearate amino acid peptide ______________________________________
Chemistries utilizing such esters have been abetted by various hydrolysis accelerators, as well as diazonium salt coupling agents. As accelerators, many alkanols have been employed, as have certain metal complexes, pyridine derivatives and imidazoles. Diazonium cations having appropriate anions ionically bound or associated therewith are well known as coupling agents for such chemistries, whereby the alcohol (phenol) formed upon hydrolysis of the ester is coupled with the diazonium salt for forming an azo dye.